(1) To develop animal, especially nonhuman primate, models that mimic human disease: We have established nonhuman primate (NHPs) models using Cynomolgus macaques for several influenza A viruses including the newly emerged H1N1 swine-origin influenza virus (SOIV), H1N1 and H3N2 seasonal influenza strains, H2N2 pandemic strain, and an H2N3 swine influenza strain. We used a recently established infection route that combined oral, intranasal, ocular and intratracheal inoculation of a total of 7x106 plaque forming units or tissue culture dose 50 (TCID50) units. In general, the models differ in clinical symptoms, disease progression and pathology. They do mimic human disease reported as a result of infection with the above mentioned influenza strains. Seasonal influenza viruses (H1N1 & H3N2) lead to either asymptomatic or fairly mild disease in Cynomolgus macaques. Shortly after infection animals show reduced food intake and occasionally mild respiratory signs. Gross pathological changes are limited to a few areas in the lungs. Histopathological investigations of those areas reveal alveolar edema and fibrin, hyaline membrane formation and type II alveolar pneumocyte hyperplasia. Animals recover quickly form the infection and clear virus within the first few days. There is limited evidence for virus shedding from mucosal membranes of the upper respiratory tract. These studies are performed with our collaborators Drs. F. DeLeo, J. Taubenberger, M. Katze and Y. Kawaoka. Compared with the seasonal strains, the pandemic H2N2 strain showed a slightly enhanced virulence with moderate clinical symptoms and respiratory signs. Lung infiltrates, gross pathology and histopathology are in general slightly enhanced compared to the infections with seasonal viruses. Animals clear the infection with a delay and fully recover. There is limited evidence for virus shedding from mucosal membranes of the upper respiratory tract. We have analyzed a total of three different strains of the newly emerged SOIV. Interestingly, we observed different degrees of clinical severity, gross pathology, lung infiltration and histology. One of the Mexican strains isolated from a moderately sick human behaved similarly to the pandemic H2N2 strain. The California isolate from a sick boy with moderate disease was more severe and the Mexico strain from a fatal case was quite severe in clinical disease and pathology. All animals survived the infection but the animals with the severe infection had not completely cleared virus at the end of the study (day 14 post infection). This clearly indicates that a variety of SOIV strains with different pathogenic potential co-circulate in the Americas and most likely worldwide. The project is performed in collaboration with Drs. M. Katze and Y. Kawaoka. Infection with the swine H2N3 virus was also more severe and was similar to the disease caused by the California strain of SOIV. Thus, this newly described swine virus can infect nonhuman primates and cause disease; it could potentially also infect humans. The work on the swine H2N3 virus is performed in collaboration with Dr. J. Richt. Overall, our findings demonstrate that influenza A virus subtypes cause disease with different degree of severity in the Cynomolgus macaque model. At least with the newly emerged SOIV circulating strains within a subtype display different pathogenic potential. (2)To identify and characterize determinants of pathogenicity in these animal models: We have started to develop a reverse genetics system for the newly emerged SOIV. In order to identify determinants of pathogenicity we used a computer-generated consensus sequence for this virus based on all reported sequences in the databases. All plasmids for the reverse genetics system have been constructed and sequence confirmed. In the next month we will start the rescue of the consensus SOIV. Subsequently, we will introduce mutations based on sequence alignments to identify determinants of pathogenicity. Rescued mutant viruses will first be characterized in vitro. For in vivo work we will focus initially on the ferret model which we will establish here based on the experience of our collaborator Dr. J. Taubenberger. Selected mutants will finally be tested for their pathogenic potential in the developed Cynomolgus macaque model. (3) To develop cross-protective vaccines and test their efficacy in the developed animal models. In order to response to a pandemic situation, cross-protective vaccines would be highly beneficial, since the development of specific homologous vaccines would take too long. Therefore, we propose to generate replication-deficient and attenuated replication-competent vaccine platforms based on different mammalian adenovirus (human, pig) and vesicular stomatitis virus, respectively. The vaccine vectors will be designed to express surface immunogens (hemagglutinin, neuraminidase), internal immunogens (nucleoprotein, matrix protein, nonstructural), or a combination of both derived from seasonal, pandemic and recently emerged influenza A viruses. The vaccine candidates will first be characterized in vitro, subsequently assessed for their immunogenic properties in mice, and finally tested for their immunogenicity and protective efficacy in a rodent model. Promising vaccine candidates will be tested for protective efficacy in ferrets and our recently developed Cynomolgus macaque model. We have started to develop the above mentioned vaccine vectors during the past months of fiscal year 2009. The project will be performed in collaboration with Dr. G. Kobinger and will continue throughout the entire fiscal year 2010 and beyond. Expectations: The studies will define determinants of pathogenicity by studying mutant influenza A viruses in vitro and in vivo (mouse, ferret, NHP models). We will further develop cross-protective vaccine platforms that could be applied in emergency response situations. Significance: A better understanding of the underlying features of highly pathogenic influenza strains and the development of cross-protective vaccines are urgently needed to prepare for the emergence and control of highly lethal influenza viruses. Therefore, the proposed studies are directly associated with the primary mandates of NIAID, NIH, namely the response to emerging/re-emerging infectious diseases. The results are expected to have direct impact on national and international public health.